The present invention relates to the introduction of DNA episomes into plant cells to silence nuclear or chromosomal genes. More particularly, this invention relates to use of geminivirus vectors to provide systemic silencing of an endogenous plant gene in a treated plant.
Gene silencing in plants typically refers to either the suppression of an endogenous gene by the introduction of a related transgene, or to the suppression of genes introduced at ectopic positions in plant genomes. Some examples of pathogen-derived host resistance to RNA viruses have been attributed to a gene silencing mechanism (Covey et al., Nature 386:781 (1997); Mueller et al. Plant J. 7:1001 (1995); Ratcliff et al., Science 276:1558 (1997); Tanzer et al., Plant Cell 9:1411 (1997). Transcriptional gene silencing has been hypothesized to involve DNA/DNA pairing, DNA methylation or heterochromatinization (Kumpatia et al., Plant Physiol. 115:361 (1997); Neuhuber, Mol. Gen. Genetics 247:264 (1995); Park et al., Plant J. 9:183 (1996)). Repeated DNA has a tendency to undergo transcriptional silencing, which may be associated with changes in chromatin structure (Meyer, Biol. Chem. Hoppe Sayler 377:87 (1996); Ye and Signer, Proc. Natl. Acad. Sci. USA 93:10881 (1996)), as well as to induce certain types of post-transcriptional silencing (Stam et al., Plant J. 12:63 (1997)). Both cytoplasmic and nuclear events have been implicated in gene silencing. Post-transcriptional gene silencing may require accumulation of a threshold level of mRNA, after which degradation of all homologous gene transcripts occurs, and may be potentiated by aberrant mRNAs. Metzlaff et al., Cell 88:845 (1997); Mueller et al., Plant J. 7:1001 (1995).
Geminiviruses are single-stranded DNA viruses that replicate through double-stranded DNA intermediates using the plant DNA replication machinery. Geminiviruses replicate in the nucleus, and foreign DNA can be stably integrated into the viral genome without significantly affecting replication or movement. Tomato golden mosaic virus (TGMV) is a bipartite geminivirus with a genome of two circular molecules, TGMV-A and TGMV-B (FIG. 1). TGMV-A replicates autonomously. The TGMV coat protein (AR1) is dispensable for replication and movement in N. Benthamiana and can be replaced with up to 800 bp of foreign DNA, which is stably maintained in the viral genome (Elmer and Rogers, Nucl. Acids. Res. 18:2001 (1990)).
A plant virus may systemically infect a plant by spreading from the initially infected cell to neighboring cells, and subsequently throughout the plant. Plant cell walls prevent the random cell-to-cell transfer of virus, but channels (plasmodesmata) in plant cell walls provide an intercellular continuum through which virus particles or viral nucleic acids may move. Viral movement via plasmodesmata is mediated by virus-encoded proteins. Citovsky et al., BioEssays 13:373 (1991). Additionally, movement of virus to parts of the plant distant from the point of initial infection can occur via companion cells and sieve elements of the phloem. However, even in systemically infected plants the distribution of the virus may not be uniform. Certain areas of the plant, even within a tissue or a structure, may contain lower or higher amounts of virus than neighboring areas.
A first aspect of the present invention is a geminivirus silencing vector comprising a geminivirus genome which contains heterologous DNA, where the heterologous DNA is identical to, or has substantial sequence similarity to, a gene endogenous to a plant or a fragment of a gene endogenous to a plant.
A further aspect of the present invention is a DNA construct comprising a geminivirus genome, where the DNA encoding the geminivirus coat protein has been replaced in part or in total with heterologous DNA identical to, or having substantial sequence similarity to, an endogenous plant gene or a fragment of an endogenous plant gene.
A further aspect of the present invention is a DNA construct comprising, in the 5xe2x80x2 to 3xe2x80x2 direction, a geminivirus origin of replication; DNA encoding proteins necessary for replication of the DNA construct; and a heterologous DNA segment identical to, or having substantial sequence similarity to, an endogenous plant gene or a fragment of an endogenous plant gene.
A further aspect of the present invention is a DNA construct comprising, in the 5xe2x80x2 to 3xe2x80x2 direction, a geminivirus origin of replication; DNA encoding proteins necessary for replication of the DNA construct; and a heterologous DNA segment identical to, or having substantial sequence similarity to, an endogenous plant gene or a fragment of an endogenous plant gene. This DNA construct is subject to the proviso that the DNA segment is not operably linked to a promoter.
A further aspect of the present invention is a method of silencing the expression of a plant gene in a plant cell, by inoculating the plant cell with a DNA construct or silencing vector as described above.
A further aspect of the present system is a method of systemically silencing expression of a plant gene in a plant, by inoculating the plant with an endogenous plant gene or a fragment of an endogenous plant gene.
A further aspect of the present invention is a method of screening isolated plant DNA for function. The method comprises preparing a DNA vector as describe above, containing a DNA segment identical to, or having substantial sequence similarity to, the isolated plant DNA or a fragment thereof. A test plant is then inoculated with the vector and allowed to grow for a period of time, then compared to a non-inoculated or sham inoculated control plant. Differences between the inoculated and control plants indicate the function of the isolated plant DNA.